Cytoprotective compounds

ABSTRACT

The present invention provides compositions and methods for protecting cells from injury due to intrinsic membrane lysis, oxidation and/or invasion by destructive agents. Even more particularly, the present invention provides compositions and methods for treating or prophylactically inhibiting phospholipase mediated injury, injury due to oxidation, and inflammation. In a very specific sense, this invention provides compositions and methods of making these compositions that are inhibitors of phospholipase.

This application is a continuation of U.S. application Ser. No.09/017,511, filed Feb. 2, 1998, now U.S. Pat. No. 6,020,510, which is acontinuation of U.S. application Ser. No. 08/632,030, filed Apr. 15,1996, now U.S. Pat. No. 5,859,271.

FIELD OF THE PRESENT INVENTION

The present invention relates to compositions and methods for protectingmammalian cells from injury due to intrinsic membrane lysis, oxidationand/or invasion by destructive agents. In particular the presentinvention relates to compositions and methods for treating againstand/or prophylactically inhibiting the injury causation. Even moreparticularly, the present invention relates to bioactive agents and theuse thereof for treating or prophylactically inhibiting phospholipasemediated injury, injury due to oxidation, and inflammation. In aspecific sense the present invention provides agents for preventingand/or treating inflammation and cell destruction in mammalian tissueand for protection and preservation of biologic material derived fromanimals, humans and plants such as food and tissue samples. In a veryspecific sense, this invention provides compositions that are inhibitorsof phospholipase and methods of making these compositions.

BACKGROUND OF THE INVENTION

The base structure of all living organisms is the cell which isstructurally defined by its membranous lipoprotein envelope. Themembranous network that holds the cell together maintains the ionicbalance and provides the receptors for hormones and neurotransmittersthat enable a cell to interact with its environment. This is pertinentto interaction with neighboring cells which enable isolated cells,tissues, or whole organisms to survive as both independent units and asparticipants in cellular interactions, in vitro and in vivo.

External factors which govern cell function, renewal, reproduction anddeath act via their effects on the phospholipid bilayer and proteins ofthe cell membrane. This controls the receptor-mediated signals and ionicfluxes which govern cell responsiveness and survival. Damage to the cellmembrane with particular emphasis on lipid peroxidation, membraneoxidation and the action of phospholipases, affects resistance toinjury, repair and host responses to environmental change and ionic andosmotic integrity.

Pathological events in a host under clinical circumstances can result incellular insult, leading to loss of membrane integrity. The events aremediated by factors which digest and destroy cell membrane and propagatean injury by producing a cascade of cell membrane changes. Byinterfering with the cascade of external and internal events involvingmembrane integrity and toxic changes which lead to cell death, injurycan be prevented, modified or reversed. This has been a major role ofanti-inflammatory agents in the past.

The most important presently used clinically effective anti-inflammatorydrugs include the corticosteroids and the non-steroidalanti-inflammatory drugs (NSAIDs). Corticosteroids inhibit the activityof cell phospholipases among other actions. NSAIDs inhibit themetabolism by cyclooxygenase of arachidonic acid released byphospholipases. These drugs act to control inflammation and to minimizecell injury by regulating the breakdown of phospholipids. These drugsalso affect the action of the products of phospholipid breakdown leadingto the formation of prostaglandins and leukotrienes which are producedin increased quantities in inflammation and promote cell dysfunction andinjury.

In addition, cellular and extracellular phospholipases may be activatedby the generation of oxygen free radicals. This can establish a damagingcycle as phospholipase activation can release free radicals which, inturn, activate more phospholipases. In this regard, free radicals areproduced from the fatty acids which are released by the action ofphospholipases and then converted to prostaglandins and leukotrienes bycyclooxygenase and lipoxygenase enzymes with oxygen free radicalproduction as a by product. Fatty acids and free radicals are known tobe prime mediators in the cascade of reactions that result in membraneinjury, cell death and inflammation. Phospholipase A₂ (PLA₂), a keyenzyme in the metabolism of phospholipid, can promote fatty acidrelease. PLA₂ may be activated by a variety of factors involvinghormonal, neural, metabolic, or immunologic pathways.

One of the hallmarks of inflammation and cell injury is the breakdown ofcellular membrane phospholipid. Phospholipids are the major structuralbuilding blocks of the cell membrane; they give rise to thebarrier-structural and functional properties of membranes and theirintegrity is crucial to normal cell responsiveness and function.Phospholipid changes in cell membrane integrity, particularly changes infatty acids at the 2 position, alter the fluidity of cell membranes,cell receptor function and the availability of cellular contents to theexternal environment. The breakdown of phospholipid membranes results inlysis of cells, produces holes in the cell membrane, affects ionchannels and membrane receptors which destroy cellular integrity andfunctional responses.

During inflammation, phospholipases, from whatever source, that arenormally under the control of natural suppressor systems, are activatedto degrade membrane phospholipid which, in turn, generates oxygen freeradicals. PLA₂ is a key enzyme which is activated in inflammation tometabolize substrate phospholipids and release free fatty acids. Thesefatty acids (i.e., arachidonate) released by PLA₂ are converted topotent biologically active metabolites, lysophospholipids,prostaglandins, and leukotrienes. These are themselves substrates forother enzymes leading to the production of thromboxanes, plateletactivating factor and other substances, with the concomitant generationof oxygen free radicals.

Phospholipases, particularly PLA₂, as membrane targeted enzymes, play animportant role since expression of their activity results in furtherproduction of inflammatory mediators leading to membrane injury whichpropagates damage within the cell itself or to adjacent tissue. Thus,the spread of injury from the initial site to contiguous or distantsites can be promoted by the activation and/or release of PLA₂.

In addition to the intrinsic membrane-related tissue breakdown via theactivation of PLA₂, phospholipases, and particularly PLA₂, are part ofthe normal defense system of the body. PLA₂ is found in human whiteblood cells (WBCs). WBCs play a role in resisting infection, but whenthese cells are mobilized to ward off injury and infection, PLA₂ isreleased from adherent and circulating WBCs and produces local tissueactivation which can increase the extent of initial injury. In addition,WBCs adhere to blood vessel walls where they release enzymes such asPLA₂. WBCs also generate free radicals such as superoxide, in largequantities, and thus promote damage to the vascular endothelium, lungalveoli or to tissue sites contiguous with WBC infiltration orconcentration. Where inflammation is found, WBCs are usually present inabundance and the WBCs adhere to vascular endothelium, with subsequentrelease and activation of PLA₂ resulting in damage to vascular integrityduring shock and ischemia. Thus, in spite of being a prime defensesystem of the body against infection, WBCs can also damage the body bypropagating injury and inflammation.

A classical description of inflammation is redness and swelling withheat and pain. Inflammation has been defined as the reaction ofirritated and damaged tissues which still retain vitality. Inflammationis a process which, at one level, can proceed to cell death, tissuenecrosis and scarring. At another level, inflammation can be resolvedwith a return to normalcy and no apparent injury or with minimalchanges, i.e., pigmentation, fibrosis or tissue thickening with collagenformation related to healing and scarring.

Microscopically, inflammation has been described as: (1) atony of themuscle coat of the blood vessel wall; (2) endothelial adherence ofinflammatory cells followed by migration of these cells from thevascular space into tissue.

The events described above are often mediated by phospholipaseactivation, followed by fatty acid release and the formation of freeradicals. Cytokines, secreted by immune cells, induce PLA₂ secretion bytheir actions on a variety of cells. Interleukin-6 stimulateshepatocytes to increase PLA₂ secretion many-fold. Interleukin-1 andtumor necrosis factor induce PLA₂ secretion by endothelial cells and bychondrocytes. Thus, immune cell products directly stimulate thehydrolysis of membrane phospholipids and production of arachidonic acidmetabolites by a variety of target cells, amplifying the inflammatoryresponse.

Alternatively, increased phospholipase activity can relate to exogenousenzyme released from infecting pathologic organisms such as viruses,bacteria, Rickettsia, protozoa, and fungi. These pathogens often possessphospholipases as factors intrinsic to their infectious activity. In thecase of Naegleria fowleri, a pathogenic amoeba with affinity for thebrain, destruction of brain membranes induced by phospholipases secretedby Naegleria can occur at sites in the brain distant from where theorganism is localized. In another example toxoplasma cannot enter thehost cell if its PLA₂ enzyme is inhibited by a specific drug. What isneeded to treat certain infections, particularly intracellularpathogens, is an effective PLA₂ inhibitor. Such an effective PLA₂inhibitor is particularly needed in cases of protozoal infections forwhich there are few effective antibiotics.

PLA₂ is also one of the major toxic components of snake venom. Bites ofcertain snakes inject venom containing PLA₂ into the wound, causingtoxic and inflammatory responses which may be lethal. What is needed areinhibitors of PLA₂ which may be administered to recipients of snakebites and bites of other animals.

Pathologic effects of phospholipases may be local, regional or systemic.These pathologic effects are governed by the phospholipase enzymereleased, the level of albumin, natural inhibitors of enzyme action, andfactors of diffusion, circulation and tissue vulnerability based onintrinsic inhibitors or the susceptibility of previously damaged oroxidized membranes or proteins to phospholipase action.

Inflammation is associated with trauma, infection and host defensereactions related to direct bacterial or virus killing by the associatedimmune responses. In general, immune responses can be both beneficial,protective or tissue damaging as can be seen in their role to promoteresistance to infection or cure of infection. Alternatively, immuneresponses may produce autoimmune phenomena that result in allergy, i.e.,asthma, urticaria, in graft versus host disease, in glomerularnephritis, in rheumatic fever, or in lupus and rheumatoid arthritis.

In regard to the current treatment of inflammation, corticosteroids areeffective anti-inflammatory agents, but must be used cautiously becausethey are powerful immunosuppressants and inhibitors of fibroblasticactivity necessary for wound healing and bone repair. In addition,corticosteroids have powerful hormonal activities and their toxic sideeffects involve interference with wound repair and bone matrixformation, sodium retention, potassium loss, bone demineralization,decreased resistance to infection, and diabetes. Corticosteroids alsohave effects on steroid formation, cataracts, blood pressure, proteinutilization, fat distribution, hair growth and body habitus.Alternatively, the clinically active NSAIDs, such as aspirin,indomethacin, ibuprofen, etc., work by inhibiting the conversion of freefatty acids to prostaglandins. The side effects of NSAIDs includegastric ulceration, kidney dysfunction and Reye's Syndrome. Metabolitesof prostaglandin can be either damaging or protective to cells dependingon the structure of the prostaglandin produced or utilizedpharmacologically and the route of administration, cell or tissueeffected.

As discussed previously, in conjunction with fatty acid release,leukotrienes are generated as part of phospholipid cell membranemediated injury produced by phospholipase activation. These leukotrienesproduced from membrane phospholipid breakdown damage tissue throughdirect toxic action, effects on ionic channels, and associated freeradical formation. Leukotrienes also damage tissue by indirect effectson vascular smooth muscle or on the vascular endothelial lining viaeffects on platelets, WBCs, or endothelial cells, or secondarily througheffects on constriction of smooth muscle. Leukotrienes are responsiblefor smooth muscle constriction leading to bronchospasm and the asthmaticattacks seen in allergy or infectious asthma. Thus, there is an ongoingsearch for leukotriene inhibitors for clinical application in thetreatment of allergy, asthma and tissue injury and inflammation.

Because the phospholipase-activated biochemical pathway for theformation of prostaglandins and leukotrienes derived from free fattyacids is branched, inhibition of one branch of this pathway, as withNSAIDs, can create an imbalance in these reactive metabolites. Thisimbalance may actually aggravate inflammation and promote cell injury asevidenced by the gastric ulcerative side effects of NSAIDs.

Due to these adverse effects of both steroids and NSAIDs, there is greatclinical interest in identifying phospholipase inhibiting agents that donot have steroidal or NSAID side effects, but like corticosteroidsmodulate the first step leading to the production of injuriousmetabolites, fatty acids and free radicals.

Free radicals, produced by white blood cells, tissue injury or metabolicprocesses, are highly reactive chemical species which, in the case oftissue injury, are most often derived from respiratory oxygen. Oxygen,while necessary for energetics of life, is also a toxin which, as thechemically related superoxide, or as peroxides, can damage tissueinstead of supporting it. Free radicals derived from oxygen are criticalto damage produced by radiation, inflammation, reperfusion tissue injuryor through excess oxygen inhalation or exposure. Free radicals are usedby white blood cells to destroy infecting organisms, but can, undercircumstances of shock, infection and ischemia, damage or destroy thetissue they were meant to protect. Free radicals, induced by radiation,oxygen exposure, chemical agents (i.e., alkylating agents, dioxin,paraquat) or white blood cell reactions may damage tissue or be involvedin mutational changes associated with aging, radiation or chemotherapyinjury, the development of cancer, and hyperimmune proliferative diseasesuch as rheumatoid arthritis. In addition, these reactive chemicalspecies can, through oxidation of proteins, enhance the vulnerability ofproteins to protease digestion.

The exact pathologic mechanisms of many skin inflammations, such asatopic dermatitis, are not clear, but probably involve inflammatorycells which can secrete or respond to PLA₂. Allergic diseases involvetissue mast cells, which can be primed or triggered by PLA₂ for therelease of their inflammatory granule contents, such as histamine. Thesecells also release additional PLA₂. What is needed are inhibitors ofPLA₂ that adequately penetrate skin after topical application andpossess prolonged anti-PLA₂ activity.

Previous published studies have demonstrated high levels of aproinflammatory PLA₂ in human herniated vertebral discs. The isolatedenzyme is toxic to dorsal root ganglion cells in culture and excisedsciatic nerve. While not wanting to be bound by this statement, it isbelieved that PLA₂ may mediate inflammation and nerve tissue damage inspinal cord injury and in sciatic nerve inflammation and may alsomediate a variety of neurological inflammatory conditions. Recently,Stephenson et al., (Neurobiology of Disease 3:51-63 (1996) have observedelevated cytosolic PLA₂ activity in brains with Alzheimer's disease.

PLA₂ also has the capability to induce severe, delayed neurotoxicitysyndrome, including extensive cortical and subcortical injury toforebrain neurons and fiber pathways, when injectedintracerebroventricularly as described by Clapp et al. (Brain Research693:101-111,1995) the entirety of which is incorporated herein byreference. We have also observed that preparations of PLA₂ andhomogenates of human vertebral disks containing extracts of the nucleuspulposus are inflammatory when injected into the mouse paw and induceedema. Edema induced by human disk homogenate is maximal between 1-3 hrsand remains so for at least 6 hrs. These results support the hypothesisthat leakage of nucleus pulposus from a herniated disk may promoteinflammation in human disk disease. Accordingly, what is needed areinhibitors of PLA₂ mediated inflammatory processes. Such inhibitorsshould alleviate the inflammation and resultant pain and discomfortassociated with disk disease and other neurological inflammatoryconditions.

Tissues that are excised from animals for subsequent transplantationinto recipients often display damage following transplantation duringreperfusion of ischemic tissue. Both ischemia and reperfusion increasePLA₂ activity and release leading to inflammatory processes with markedactivation of the vascular endothelium. These processes decrease theprobability of successful transplantation thereby increasing theincidence of rejection and the need for additional immunosuppressivetherapy. Such problems greatly increase morbidity and mortality,increase the costs of treatment and insurance, and result in lost timeat work. What is needed are drugs that will inhibit PLA₂ activity andenhance tissue preservation before transplantation thereby decreasingischemia reperfusion injury.

Infections caused by parasites constitute a major public health problemthroughout the world for humans and animals, annually resulting insignificant incidence of disease, suffering and death. Parasites such asthose that cause malaria and other protozoal parasites of animals andhumans are especially troublesome. We have found that the PLA₂inhibitor, quinacrine (mepacrine), significantly reduced molting oflarval forms of an animal filarid. What is needed are new compounds thateffectively inhibit PLA₂ activity for application to parasites such asthose causing malaria and other protozoal parasites injurious to animalsand humans.

A previous study by Clay et al. (Third International Congress:Eicosanoids & Other Bioactive Lipids in Cancer, Inflammation, &Radiation Repair, Abstract #162) reported that the product of PLA₂activation, 1-acyl lysophospholipid, which affects membrane fluidity,accumulates in stored blood and may be taken up by white blood cells(WBCs) and used to make platelet activating factor (PAF) thereby“priming” WBCs during storage and promoting injury during subsequenttransfusion. It has been suggested that increased PLA₂ activity mayperturb cells in storage. What is needed are compounds that protectblood cells and other cells during storage so that these cells will notcause problems when utilized.

Accordingly, what is needed are compounds and methods of using thesecompounds which provide protection against the deleterious effects ofPLA₂ activation. These compounds should be capable of inhibiting PLA₂,thereby decreasing the PLA₂ metabolites which are substrates for thecyclooxygenase, 5-lipoxygenase, 12-lipoxygenase, and other enzymaticpathways which lead to formation of cyclic endooperoxides,prostaglandins (such as prostacyclin and thromboxane), leukotrienes, andplatelet activating factor. These compounds should decrease inflammatoryprocesses and free radical production in a variety of tissues and cells.They should be capable of being administered in vivo (topically, orally,by injection and through other means), ex vivo and in vitro and shouldalso exhibit low or no toxicity. These compounds should displaydifferent solubilities in lipid and aqueous systems depending on themode of application and the desired target.

SUMMARY OF THE INVENTION

The present invention provides both lipid and/or water soluble compoundsthat are PLA₂ inhibitors having antioxidant properties and/orantiinflammatory properties. This invention provides bioactive compoundswhich are oligomers (dimers, trimers and tetramers, etc.) of fattymoieties that inhibit PLA₂ activity. The terms dimer, trimer, tetramerand pentamer as used throughout the present description define thenumber of fatty moieties present in the particular molecule. That is tosay a dimer has two fatty moieties, a trimer has three, a tetramer hasfour, a pentamer has five, etc. The compounds of the present inventionpossess at least one double bond to enhance their anti-inflammatory andcytoprotective or tissue protective effects.

The compounds of the present invention may be used for treating orprophylactically inhibiting phospholipase mediated injury and/or injurydue to oxidation. In a specific sense, the present invention providesagents for preventing and/or treating inflammation and cell destructionin mammalian tissue and for protection and preservation of biologicmaterial such as cells, tissues, organs and fluids obtained from animalsand humans. The present invention also provides agents for protectionand preservation of food obtained from animals and plants, and forcellulose products and wood products obtained from plants. The compoundsof the present invention protect phospholipid cell membranes, andproteins from the effects of oxidative injury or aging. These compoundsof the present invention also inhibit free radical reactions and therebystabilize proteins for maintenance of biologic half-life, anti-coagulantactivity, and food preservation.

More specifically, the present invention provides pharmacologicallyactive, anti-phospholipase compounds. These compounds are soluble and/ordispersible in a suitable carrier. The compounds may exist as oligomerssuch as dimers, trimers, tetramers etc., or as combinations thereof. Inaccordance with the present invention, the compounds have at least twofatty moieties and contain at least one unsaturated double bond. Eachfatty moiety may take a variety of forms, may possess the same ordifferent functional groups, may be a different length. In one preferredform, the compounds of the present invention may have an acid group orany salt form thereof. The compounds of the present invention may alsobe present in ionized form.

Accordingly, an object of the present invention is to providecompositions that inhibit the activity of the enzyme PLA₂.

It is a related object of the ,present invention to decrease levels ofproducts of the enzymes cyclooxygenase, 5-lipoxygenase, 12-lipoxygenase,prostacyclin oxycyclase, thromboxane synthase, and prostaglandinisomerase pathways by inhibiting the activity of PLA₂.

Another object of the present invention is to provide methods ofsynthesizing these compositions that inhibit the activity of PLA₂.

It is also an object of the present invention to provide methods oftreating conditions associated with PLA₂ activity.

Yet another object of the present invention is to provide a compositionand treatment for oxidative and free radical damage to cells, tissuesand organs in vitro and in vivo.

Another object of the present invention is to provide methods ofapplying an effective amount of the compositions to treat inflammation.

Another object of the present invention is to provide a composition andtreatment for inflammatory processes.

It is also an object of the present invention to provide oral andtopical treatments for arthritis with the compositions of the presentinvention.

Yet another object of the present invention is to provide treatments forpain using the composition of the present invention.

Another object of the present invention is to provide oral and topicaltreatments comprising administration of an effective amount of thecompositions of the present invention, for a variety of conditions, suchconditions including, but not limited to, the following: reflexsympathetic dystrophy; inflammation of the central and peripheralnervous system, diseases related to inflammation of the nervous systemincluding, but not limited to, Alzheimer's disease, inflammation ofspinal nerves, autonomic nerves and cranial nerves; inflammatoryradiculopathy; back pain including low back pain; myo-fascial painsyndromes; inflammation of the connective tissues, including meninges,inflamed and diseased facet joints, herniated disks, diseased disks,torn and injured annulus fibrosis, diseases of the joints, ligaments,cartilage and synovial membranes; mastocytosis; shock including septicshock, anaphylactic shock, anaphylactic shock resulting fromradiocontrast administration, and shock resulting from bacterialinfections; bacterial infections; uremia; autoimmune disorders;parasitic infections including, but not limited to, malaria;inflammation including allergic inflammation; skin inflammation,itching, and other dermatologic disorders due to allergic reactions, dryskin, erythema, solar, nuclear and other forms of radiation, windburn,acne, psoriasis, eczema, reactions to chemicals and toxins, contactdermatitis, and reactions to plants including, but not limited to,poison ivy, poison oak, poison sumac; bites of insects including, butnot limited to, mosquitos, fire ants, chiggers, ticks, bees, spiders,fleas and flies; bites of reptiles, especially venomous reptiles,amphibians, and other animals; contact with various animals with venomon their skin such as poisonous frogs; pruritis associated with localdermatologic or systemic disease; prevention of tissue ischemiaincluding tissue in vivo and tissue destined for transplantation,prevention of ischemia-reperfusion injury, prevention ofischemia-reperfusion injury in the setting of resuscitation fromhypovolemic shock, renal ischemia, myocardial infarction, angina, andcardiac ischemia; endothelial inflammation, cardiotoxicity associatedwith administration of anti-cancer compositions, inhibition of coronaryor cerebral restenosis following angioplastic or other vascularprocedures, inhibition of platelet activity, especially in vesselsfollowing various procedures such as angioplasty and after insertion ofcatheters, shunts and other devices, inhibition of thrombin-activatedplatelet aggregation; pulmonary diseases including, but not limited to,asthma, cystic fibrosis, inflammation of the lungs secondary to ischemiaof the gastrointestinal system, adult respiratory distress syndrome, andother allergic and inflammatory reactions of the pulmonary systemincluding inflammation of the tissues of the upper respiratory system,allergic rhinitis, and respiratory distress syndrome; inflammation ofthe gastrointestinal system including, but not limited to, Crohn'sdisease, eosinophilic gastroenteritis, peritonitis, ulcerative colitis,ulcers of the small bowel and stomach, esophagitis, inflammation of thestomach, inflammatory bowel disease; ocular inflammation; preservationof whole blood; preservation of tissues, cells, and organs fortransplantation; and protection of mitochondria.

Another object of the present invention is to provide a method ofapplying an effective amount of the compositions of the presentinvention through injection, topical, oral, or aerosol administration,for the treatment of inflammation resulting from the bites of insects,reptiles, amphibians, and other animals, especially venomous animals,such as venomous snakes.

Another object of the present invention is to provide a composition andmethod for inhibition of platelet function.

It is an object of the present invention to provide a composition forprevention and treatment for acute and chronic rejection of transplants,and for treatment of graft-vs-host disease and autoimmune diseases.

It is another object of the present invention to provide a compositionfor the treatment of neoplastic disease.

It is another object of the present invention to provide an easy to usetopical therapeutic composition and topical treatment for various formsof arthritis and other inflammatory diseases, including, but not limitedto, rheumatoid arthritis, inflammatory arthropathies, osteoarthritis,gout, and lupus.

It is another object of the present invention to provide a compositionand treatment for parasitic infections including, but not limited to,toxoplasmosis, malaria, Naegleria fowleri, Dilofilaria immitis,nematodes, and pathogenic protozoans such as toxoplasma gondii,falciparum malaria, amebiasis, amoeba, and cryptosporidia.

It is another object of the present invention to provide the enhancedrange of motion and reduced pain provided to patients with reflexsympathetic dystrophy following topical or oral application of thecompositions of the present invention.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following applications are hereby incorporated by reference in theirentirety: application Ser. No. 08/467,690 filed Jun. 6, 1995;application Ser. No. 08/475,335 filed Jun. 7, 1995; application Ser. No.08/010,456 filed Jan. 27, 1993; application Ser. No. 07/839,780 filedApr. 1, 1992; application Ser. No. PCT/US90/04615 filed Aug. 16, 1990;application Ser. No. 07/399,941 filed Aug. 29, 1989; application Ser.No. 07/256,330 filed Oct. 11, 1988; application Ser. No. 07/156,739filed Feb. 18, 1988; and application Ser. No. PCT/US87/00408 filed Feb.24, 1987.

Cis-unsaturated, but not saturated fatty acids, inhibit in vitro PLA₂activities derived from human platelets and human polymorphonuclearleukocytes (PMNs). PLA₂ activity is inhibited by oleic, linoleic, andarachidonic acids to approximately the same extent indicating that thepresence of a single cis-double bond is as inhibitory as multiplecis-double bonds. In contrast, fatty acids containing trans-double bondsor methyl esters of cis-unsaturated fatty acids are less inhibitory ofPLA₂ activity. Thus, it is hypothesized that the preferred structuralcharacteristics for inhibition of in vitro PLA₂ activity by unesterifiedfatty acids include at least one double bond. Oleic acid inhibits invitro PLA₂ activity due to the presence of a single double bond at theC-9 position. Oxidation of the sarcoplasmic reticulum of muscle and ofphospholipid membranes predisposes them to phospholipase degradation.Phospholipid membranes that have been oxidized at particular sites mayappear intact and maintain functional activity, but their oxidationmakes them vulnerable to degradation and destruction by PLA₂ or otherphospholipases from endogenous or exogenous sources.

The observations illustrating the enhanced vulnerability of phospholipidmembranes to phospholipase following oxidative and free radical mediatedchanges in cell membranes and/or cisunsaturated fatty acids have beenemployed in accordance with the present invention in the design of novelanti-inflammatory and cytoprotective agents. The present invention thusprovides a biochemical and synthetic organic approach to controlling theexpression of PLA₂ enzymes which is vital to the maintenance of membranestructure.

Although not wanting to be bound by the following hypothesis, it isbelieved that the number of available methylene interrupted unsaturateddouble bonds is directly related to the susceptibility of fatty acids tooxidation. This governs the ability of unsaturated fatty acids to act asanti-oxidants. This property, in conjunction with the anti-PLA₂ activityof the fatty moiety compounds of the present invention, markedly expandsthe scope of the anti-inflammatory and cytoprotective activity of thenew agents disclosed herein. It is the property of the dual action ofthose compounds, i.e., their action as PLA₂ inhibitors with varyinganti-oxidant activity, that provides the spectrum of anti-inflammatoryactivity in model systems that have direct applicability tocytoprotection and the control of inflammation and pathophysiology.

A nonfunctional alkyl chain or group is known as a hydrocarbon groupbecause it contains only C—C and C—H single bonds. A nonfunctional alkylchain therefore contains the maximum possible number of hydrogens percarbon which may be represented as —C_(n)H_(2n+1). A functionalizedalkyl chain or group has substituted for one or more hydrogens on thealkyl chain, one or more atoms or groups of atoms that havecharacteristic chemical behavior. These atoms or groups of atoms thathave characteristic chemical behavior are also known as functionalgroups. Included in these functional groups are C═C, OH, COOH, SO₃H,PO₃H, NH₂, —O—, and halides,

In summary, a single double bond in a fatty moiety compound issufficient to inhibit PLA₂ activity in vitro and in situ. The additionof multiple double bonds provides the additional value of an increase inpotent anti-oxidant activity along with PLA₂ inhibitory action. Thepresent invention thus provides compounds characterized by bothanti-PLA₂ and varying anti-oxidant activity to maximize theanti-inflammatory and cytoprotective action which is the key to theclinical value of the compounds of the present invention.

In addition to inhibiting PLA₂ activity, the anti-oxidant action ofthese compounds protects proteins that become increasingly vunerable toattack by proteases due to oxidation. Thus, the cytoprotective PLA₂inhibitors of the present invention, which have anti-oxidant activity aswell, have value both in stabilizing membrane phospholipid and ininhibiting or preventing protein degradation or denaturation. Thissuggests that the compounds of the present invention act to minimizeinflammation at its onset and will also interrupt the inflammatoryprocess in progress.

The compounds of the present invention block arachidonic acid releasefrom human polymorphonuclear cells and endothelial cells, a reactionmediated by cellular and secretory PLA₂ activity. Thus, the compounds ofthe present invention are potent, reversible PLA₂ inhibitors, and, assuch, these agents inhibit the proinflammatory response of the human PMNand other inflammatory cells by inhibition of cellular and secretoryPLA₂ activity. In addition, the compounds of the present inventioninhibit, to various extents, the free radical activity in cells andtissues involved in the inflammatory process.

In a general sense, the present invention provides pharmacologicallyactive, anti-phospholipase compounds. Preferably the compounds of thepresent invention are water or lipid soluble antioxidants. The preferredcompounds have at least two fatty moieties and contain at least oneunsaturated double bond. The fatty moieties may be different from eachother in several features including, but not limited to, chemicalcomposition, functional groups, the degree of unsaturation, and thelength of the hydrocarbon chain. The compounds may also have at leastone organic group, one active acid group or any salt form or ionizedform thereof. The invention contemplates a variety of configurationsincluding oligomers such as dimers, trimers, tetramers, and combinationsthereof. Several of these compounds provided in accordance withprinciples and concepts of the present invention may be prepared asoutlined in the following specific examples.

The compounds of the present invention are not generally hydrolyzed bypancreatic enzymes and are different from glycerol-based compounds interms of their chemistry and metabolism. For example, we have observedthat two compounds of the present invention; PX-13 and PX-18, areresistant to degradation in vitro by the commercially availablepancreatic enzyme preparation, pancreatin, obtained from Sigma ChemicalCompany (St. Louis, Mo.). The resistance of PX-13 and PX-18 tometabolism by pancreatic enzymes supports their stability after oraladministration.

The oligomers (dimers, trimers, tetramers, pentamers, etc.) ofunsaturated fatty acids and the other compounds having at least oneunsaturated straight chain fatty radical as described above, affectfundamental membrane phospholipid reactions of phospholipase-induceddegradation and free radical peroxidation. The data set forth hereinconfirm with experimental results that these compounds are potentanti-inflammatory and cytoprotective agents.

The appropriate dosage of an effective amount of the unsaturated fattymoiety compounds of the present invention for treatment of mammalsincluding humans, against phospholipase mediated injury and/orinflammation should be in the range of approximately 1 to 75 mg per kg(mg/kg) of body weight and preferably approximately 2 to 50 mg/kg ofbody weight, with a more preferable range of approximately 10 to 40mg/kg of body weight, when the compound is administered orally orintraperitoneally (IP). When administered intravenously the dosageshould be approximately 50% of the oral or IP dosage to achieve the samelevel of the drug in the blood. The described dosage is also beappropriate for prevention of human platelet aggregation or bloodclotting. As is known to those skilled in the art, therapeutic dosesexpressed in terms of amounts per kilogram of body weight or surfacearea may be extrapolated from mammal to mammal, including to humanbeings. The compounds of the present invention may also be administeredin an aerosol manner, such as an intranasal spray to treat inflammationof the nasal cavities, nasopharynx and adjacent regions or as inhaledformulations to treat inflammation of the upper and lower respiratorysystem.

The compositions of the present invention may be formulated foradministration in any convenient way by analogy with other topicalcompositions adapted for use in mammals. These compositions may be usedin any conventional manner with the aid of any of a wide variety ofpharmaceutical carriers or vehicles. The compounds of the presentinvention described above can be provided as pharmaceutically acceptableformulations using formulation methods known to those of ordinary skillin the art. These formulations can be administered by standard routes.In general, the combinations may be administered by the topical,transdermal (including ionophoretic administration), buccal, oral,rectal or parenteral (e.g., intravenous, subcutaneous or intramuscular)route. In addition, the combinations may be incorporated intobiodegradable polymers allowing for sustained release of the compound,the polymers being implanted in the vicinity of where drug delivery isdesired, for example, at the site of a tumor. The biodegradable polymersand their use are described, for example, in detail in Brem et al., J.Neurosurg. 74:441-446 (1991).

A pharmaceutically acceptable solvent is one which is substantiallynon-toxic and non-irritating under the conditions used and may bereadily formulated into any of the classical drug formulations such aspowders, creams, ointments, lotions, gels, foams, aerosols, solutionsand the like. Particularly suitable solvents include, but are notlimited to, water, ethanol, acetone, glycerin, propylene carbonate,dimethylsulfoxide (DMSO), and glycols such as 1,2-propylene diol, i.e.,propylene glycol, 1,3-propylene diol, polyethylene glycol having amolecular weight of from 100 to 10,000, dipropylene glycol, etc. andmixtures of the aforementioned solvents with each other.

A topical cream may be prepared as a semi-solid emulsion of oil in wateror water in oil. A cream base formulation by definition is an emulsion,which is a two-phase system with one liquid (for example fats or oils)being dispersed as small globules in another substance (e.g., aglycol-water solvent phase) which may be employed as the primarysolvent. The cream formulation may contain fatty alcohols, surfactants,mineral oil or petrolatum and other typical pharmaceutical adjuvantssuch as anti-oxidants, antiseptics, or compatible adjuvants. A typicalcream base formulation is as follows:

Water/glycol mixture (15% or more glycol) 50-99 parts by weight FattyAlcohol    1-20 Non-ionic Surfactant    0-10 Mineral Oil    0-10 TypicalPharmaceutical    0-05 Adjuvants Active Ingredients 0.0001-10

A “classical” ointment is a semi-solid anhydrous composition which maycontain mineral oil, white petrolatum, a suitable solvent such as aglycol and may include propylene carbonate and other pharmaceuticallysuitable additives such as surfactants, for example Span and Tween, orwool fat (lanolin), along with stabilizers such as antioxidants andother adjuvants as mentioned before. Following is an example of atypical “classical” ointment base:

White Petrolatum 40-94 parts by weight Mineral Oil    5-20 GlycolSolvent    1-15 Surfactant    0-10 Stabilizer    0-10 Active Ingredients0.0001-10

Additionally, the compounds may be formulated as oral, parenteral,subcutaneous, intravenous, intraarticular, intramuscular,intraperitoneal, intralesional and otherwise systemic compositions.Depending on the intended mode, the compositions may be in the form ofsolid, semi-solid, or liquid dosage forms, such as, for example,tablets, suppositories, pills, capsules, powders, liquids, suspensions,or the like, preferably in unit dosage forms suitable for singleadministration of precise dosages.

The compositions will include a conventional pharmaceutical carrier orexcipient and a therapeutically effective amount of a compound of thepresent invention and, in addition, may include other medicinal agents,pharmaceutical agents, carriers, adjuvants, etc.

The amount of active compound administered will, of course, be dependenton the human or animal subject being treated, the severity of theaffliction, the manner of administration and the judgment of theprescribing clinician.

Typical compositions contain approximately 0.01-95% by weight of activeingredient, with the balance one or more acceptable non-toxic carriers.The percentage of active ingredient, will, of course, depend upon thedosage form and the mode of administration.

For solid compositions, conventional non-toxic solid carriers including,for example, pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharin, talcum, cellulose, glucose,sucrose, magnesium carbonate, and the like may be used. The activecompound as defined above may be formulated as suppositories using, forexample, polyalkylene glycols and propylene glycol, as the carrier.Liquid pharmaceutically administerable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier, such as water,saline, aqueous dextrose, glycerol, ethanol, and the like, to therebyform a solution or suspension. If desired, the pharmaceuticalcomposition to be administered may also contain minor amounts ofnontoxic auxiliary substances such as wetting or emulsifying agents, pHbuffering agents and the like, for example, sodium acetate, sorbitanmonolaurate, triethanolamine, sodium acetate, triethanolamine oleate,etc. Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art, for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thedition, 1975. The composition or formulation to be administered will,in any event, contain a quantity of the active compound(s) in an amounteffective to alleviate the symptoms of the subject being treated.

For oral administration of the compounds of the present invention, apharmaceutically acceptable non-toxic composition is formed by theincorporation of any of the normally employed excipients, such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium,carbonate, and the like. Such compositions take the form of solutions,suspensions, tablets, pills, capsules, powders, sustained releaseformulations and the like. Such compositions may contain 2%-95% activeingredient, preferably 5%-90%.

Parenteral administration is generally characterized by injection,either subcutaneously, intramuscularly or intravenously. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions, solid forms suitable for solution or suspension in liquidprior to injection, or as emulsions. Suitable excipients are, forexample, water, saline, dextrose, glycerol, ethanol or the like. Inaddition, if desired, the pharmaceutical compositions to be administeredmay also contain minor amounts of non-toxic auxiliary substances such aswetting or emulsifying agents, pH buffering agents and the like, such asfor example, sodium acetate, sorbitan monolaurate, triethanolamineoleate, etc.

Ointments for topical application may be prepared by incorporatingapproximately 0.1 to 10% of the compound as an oil or salt form into anointment base containing emulsifying agents such as stearic acid,triethanolamine and/or cetyl alcohol. The formulation may also includeingredients such as glycerol, petrolatum, water and preservatives asrequired.

Water based lotions may contain the compounds of the present inventionas an oil or solid in amounts ranging from approximately 0.1% to 5.0% byvolume. Such lotions may contain glycerine and/or bentonite assuspending agents as is well known in the art. The present invention mayalso be incorporated into creams.

The compounds may also be incorporated into classical (one or two phase)or non-classical (aqueous emulsion) aerosol formulations. Suchformulations include the compounds and an appropriate propellant carrierin which the compounds are dissolved or dispensed. In the classical formthe active ingredients are generally used as an oil dispersion or insolution in an organic solvent such as ethanol. In the non-classicalform the active ingredient is dissolved in water. In each case theconcentration of the active ingredient in the carrier may be aboutapproximately 0.1 to 10% by weight or volume.

Of particular advantage is the fact that the unsaturated straight chainfatty moiety compounds described above function pharmacologically at thesite of inhibitory action for the arachidonate cascade andpreferentially affect stimulus-induced mobilization of arachidonate.Inhibition of PLA₂ depresses the production of both prostaglandins andleukotrienes in stimulated or inflamed cells. Importantly, the compoundsdescribed above have a much more pronounced effect on stimulus-induced,than on controlled release of arachidonate indicating a selective effecton the former. Moreover, when phospholipids are peroxidized, the polymercompounds described above are capable of inhibiting the degradation ofsuch lipids by lysosomal phospholipase C, indicating that thesecompounds can protect already damaged (oxidized) membranes.

Thus multiple actions are responsible for the anti-inflammatory activityof the fatty moiety compounds of the present invention, and on the basisof inflammatory models, it is evident that these compounds caneffectively rival or replace both currently available steroids andNSAIDs in the treatment of inflammation, making the fatty moietycompounds of the present invention candidates for clinical applicationand usefulness in localized and systemic injury and disease.

The fatty moiety compounds described above, by protecting lipidmembranes and possessing anti-oxidant activity, are potent anti-oxidantsfor preservation, not only of living cells and tissues, but their actionmakes them effective as preservatives of biological materials fromanimal, human or plant origin, and as preservatives of chemical agentssubject to oxidative injury. For purposes of protecting and preservingbiological materials subject to oxidation injury, the fatty moietycompounds of the present invention may be used at concentrations ofapproximately 0.1 to 100 μM. These molarities are calculated as themolarity that would be obtained if the drug were dissolved in a weightof water which is the same as the weight of the biological material tobe preserved. For example, in vitro, anti-oxidant and/oranti-phospholipase applications, concentrations of from aboutapproximately 0.1 to about 500 μM should be effective.

This invention is further illustrated by the following examples, whichare not to be construed in any way as imposing limitations upon thescope thereof. On the contrary, it is to be clearly understood thatresort may be had to various other embodiments, modifications, andequivalents thereof which, after reading the description herein, maysuggest themselves to those skilled in the art without departing fromthe spirit of the present invention and/or the scope of the appendedclaims.

EXAMPLE 1 Ricinoleic Acid Related Compounds

In one preferred form, the compound may be a derivative of ricinoleicacid having a structural configuration as set forth below

where R₁ is an alkyl group or acyl group, which may be functionalized ornon-functionalized, which includes an active acid group or salt formthereof, and R₂ is an alkoxy or alkylamino group which includes one ofthe fatty moieties. More particularly, in the foregoing compound, R₂ maybe

wherein n is an integer from 1 to 18, m is an integer from 1 to 4, x isan integer from 0 to 12, and Y is —O— or —NH—, or —NR— wherein R is afunctionalized or non-functionalized alkyl group of 1 to 6 carbons.

In one case the R I moiety may be obtained by esterification of the12-position hydroxy group of ricinoleic acid with an acid group of adivalent acid such as sebacic acid, fumaric acid, maleic acid, oxalicacid, succinic acid, or organic moieties including, but not limited to,ethylenediamine tetraacetic acid (EDTA) and its analogs and derivatives,and the R₂ moiety may be obtained by esterification of the 1-positioncarboxy group of ricinoleic acid with the hydroxyl group of anotherricinoleic acid molecule or of some other fatty alcohol such as, forexample, oleyl alcohol, linoleyl alcohol, linolenyl alcohol, arachidonylalcohol or cis-5, 8, 11, 14, 17-eicosapentaenyl alcohol. Alternatively,the R₂ moiety may be obtained by amidification of the 1-position carboxygroup of ricinoleic acid with the amine group of an unsaturated fattyamine such as oleyl amine, linoleyl amine, linolenyl amine, arachidonylamine or cis-5, 8, 11, 14, 17-eicosapentaenyl amine.

EXAMPLE 2

Alternatively, the compounds of the present invention may have thegeneric structural formula as set forth below;

however, in this case R₁ may be an alkyl group which may befunctionalized or non-functionalized and includes an active acid groupor one of the fatty moieties, and R₂ may be either a hydroxy group or analkoxy group which includes fatty moieties. In this alternative case,when R₂ is a hydroxy group, R₁ may be derived by esterification of the12-position hydroxy group of the ricinoleic acid, with, for example, theacid group of oleic acid, linoleic acid, linolenic acid, arachidonicacid or cis-5, 8, 11, 14, 17-eicosapentaenoic acid. Thus, in this case,R₁ may be a fatty moiety having one of the following configurations:

where n is an integer from 1 to 18, m is an integer from 1 to 4, x is aninteger from 0 to 12, and R is H a fatty moiety or an alkyl group whichmay be functionalized or non-functionalized.

The invention contemplates compounds of a variety of configurationsincluding, for example oligomers including dimers, trimers, tetramersand combinations thereof. Dimers of ricinoleic acid are prepared forexample, by esterifying the 12-position hydroxy groups of two moleculesof ricinoleic acid with the carboxy groups of a diacid such as sebacicacid, fumaric acid, maleic acid, oxalic acid or succinic acid.

A trimer of ricinoleic acid is prepared, for example, by esterifying the12-position hydroxy groups of three molecules of ricinoleic acid withthe carboxy groups of a tri-acid such as cis-aconitic acid.

A tetramer of ricinoleic acid is prepared, for example, by esterifyingthe 12-position hydroxy groups of four molecules of ricinoleic acid withthe carboxy groups of a tetra-acid such as ethylenediaminetetraaceticacid.

Various compounds may be linked together by esterification through oneof the free acid groups. Thus, the acid groups may be converted to acidchloride groups and reacted with hydroxy or amine groups of a divalentcompound.

In each case, the compounds of the present invention include at leastone unsaturated bond and at least two fatty moieties. Desirably, eachcompound may also include at least one active acid group or any ionizedform or salt form thereof. Some of the preferred acid groups include butare not limited to sulfonyl, sulfonate, phosphoryl, phosphonate,carboxyl and carboxylate.

EXAMPLE 3 Synthesis of N,N-Bis(oleicacid-12-oxycarbonylmethyl)-N-N′bis(carboxylmethyl) ethylenediamine

This compound is represented by the following structural configuration:

To 6.22 g (20.8 mmol) of ricinoleic acid and 4.00 g of triethylamine in150 mL THF was added 2.31 g (9.0 mmol) of ethylenediamine tetraaceticacid dianhydride. After stirring 15 min at 40° C., 20 mL of acetonitrilewas added, since ethylenediamine tetraacetic acid dianhydride was notdissolved completely. The solution was stirred for 72 h under reflux andgradually turned yellow during this time. The solvent was removed underreduced pressure. The mixture was suspended in 100 mL of ether andextracted twice with 50 mL of saturated aqueous sodium chloride. Afterdrying the ether solution, the solvent was removed under reducedpressure. The residue was suspended in methanol and filtered. Thesolvent was removed under reduced pressure. The residual oil waspurified by chromatography on silica gel, eluted with a gradient ofacetone/methanol (70:30 v/v to 0:100 v/v). N,N-bis(oleicacid-12-oxycarbonylmethyl)-N-N′-bis(carboxylmethyl) ethylenediamine wasisolated as a yellow solid with a yield of 1.23 g (14%). This compoundis readily soluble in water.

N,N-Bis(oleic acid-12-oxycarbonylmethyl)-N-N′bis(carboxylmethyl)ethylenediamine exhibits an EC₅₀ value of 5 to 10 μM in the inhibitionof PLA₂ activity.

EXAMPLE 4 1,3-Bis(12-hydroxyoleoylamino)-2-hydroxypropane

This compound is represented by the following structural configuration:

To a solution of 12.60 g (31.9 mmol) of ricinoleic acidN-hydroxysuccinimide ester and 1.8 g (20 mmol) of1,3-diamino2-hydroxypropane in 125 mL of methylene chloride was added acatalytic amount of sodium bicarbonate. The suspension was stirred for48 h at 35° C. The mixture was filtered and the solvent was removedunder reduced pressure. The oil was suspended in water and extracted 3times with methylene chloride. The solvent was removed and the yellowheavy viscous oil was dried in vacuo. The yield was 930 mg.

EXAMPLE 5 1,3-Bis(oleoylamino)-2-hydroxypropane

This compound is represented by the following structural configuration:

To a solution of 8.60 g (95.4 mmol) of 1,3-diamino-2-hydroxypropane in400 mL of water was added 200 mL of methylene chloride. The emulsion wascooled to 0° C. and 85.0 g (280 mmol) of 85% oleoyl chloride were added.The mixture was stirred vigorously for 1 h at 0° C. and for 1 h at roomtemperature. The resulting precipitate was recovered by filtration andwas dried in vacuo. Reprecipitation from ethanol provided a white solidwith a yield of 31.10 g (53%) and a melting point of 91-92° C.

EXAMPLE 6 1,3-Bis(oleoylamino)-2-propyl succinic acid monoester

This compound is represented by the following structural configuration:

To a solution of 505 mg (0.817 mmol) of1,3-bis(oleoylamino)-2-hydroxypropane and 140 mg (1.40 mmol) of succinicanhydride in 20 mL of acetonitrile and 20 mL of THF was added 5 mL oftriethylamine. The suspension was stirred at 35° C. After 15 minsuccinic anhydride was completely dissolved. The solvent was removedunder reduced pressure after 48 h. The yellow oil was suspended in waterand extracted three times with ether. The solvent was removed underreduced pressure and the yellow oil 1,3 bis(oleoylamino)-2-propylsuccinic acid monoester was dried in vacuo. The yield was 380 mg (65%).

In addition to the foregoing compounds, some of which comprisederivatives of ricinoleic acids, additional compounds are includedwithin the broad scope of the present invention. Some of these compoundsare defined structurally by the following Examples.

EXAMPLE 7

One such class of compounds is defined by the following generic formula,

wherein X is an organic or inorganic anionic moiety such as bicarbonate,acetate, citrate, succinate, p-toluenesulfonate (Example 13), or halidesuch as chloride, fluoride, bromide or iodide, or phosphate, sulfate andother pharmaceutically acceptable anions; wherein R₁ is —CH₂—O—R, ahydrogen, or an alkyl group or chain which may be functionalized; andwherein the R groups may be the same or different and each R group is afatty moiety. In this form of the present invention the R groups mayhave the following structural formulation:

where n is an integer from 1 to 18, m is an integer from 1 to 4, x is aninteger from 0 to 12, and the R groups may be the same or different.

A method for preparing a particularly preferred compound having theforegoing structural configuration, p-toluene sulfonic acid salt oftristrioleate, is described in Example 13.

EXAMPLE 8 Generic TES Related Compounds

Alternatively, the compounds of the present invention may have thegeneric formula:

In this structure, A comprises H, OH, a sugar moiety, an ether, anester, an amide or NH₂, or an acid or salt thereof. Some of thepreferred acids that may be substituted for A include but are notlimited to COOH, SO₃H or PO₃H.

B is a connecting group selected from the group consisting of C,—(CH₂)_(n)C—, N⁺, and (CH₂)_(n) N⁺ wherein n is an integer from 1 to 24,and the —(CH₂)_(n) chain may be functionalized or non-functionalized.

C₁, C₂, C₃ and C₄ are connecting groups selected from the group—(CH₂)_(n)— where n is an integer from 1 to 24, wherein the —(CH₂)_(n)—chain may be functionalized or nonfunctionalized. C₁, C₂, C₃ and C₄ mayalso be selected from the group consisting of poly(ethylene oxide) ofthe formula (CH₂—CH₂—O)y wherein y is an integer from 1 to 12. C₁, C₂,C₃ and C₄ may be the same or different.

D₁, D₂ and D₃ are fatty chains consisting of fatty acid esters of theform CH₃(CH₂)_(n) COO or fatty acid amides of the form CH₃(CH₂)_(n) CONHwherein n is an integer from 0 to 32. At least one of the fatty chainsis unsaturated at one or more positions, and D₁, D₂ and D₃ may be thesame or different with respect to length and degree of unsaturation. D₁,D₂ or D₃ may also be H provided no more than one of D₁, D₂ and D₃ is Hin any one compound. In the generic formula listed above, the fatty acidchains of the molecule may be comprised of (CH₂)_(n) wherein n is aninteger from 1 to 32. These fatty acid chains may each be unsaturated atone or more sites and may be of different lengths from 1 to 32 carbonatoms.

E is H, or is the same as A-C₄, or is a fatty acid amide of the formCO(CH₂)_(n)CH₃ or CO(CH₂)_(n)COOH where n is an integer from 0 to 24.The alkyl (CH₂)_(n) chain may be functionalized or nonfunctionalized.

F is selected from the group consisting of N, NR, P, P═O, CH or CR,wherein R is an alkyl chain of 1 to 6 carbons which may befunctionalized or non-functionalized.

More specifically, the compounds may also have the formula.

wherein the R groups may be the same or different and each R is a fattymoiety as set forth below,

wherein n is an integer from 1 to 18, m is an integer from 1 to 4, and xis an integer from 0 to 12.

In this form of the present invention Z represents a C₁ to C₅ aliphaticmoiety, which may be functionalized or non-functionalized, and Arepresents an organic acid moiety or salt form thereof or any organicacid radical. It is to be understood that the acidic groups and the NHgroups in the generic structure described above may be present inionized form. Some of the preferred acid groups that may be substitutedfor A include but are not limited to COOH, SO₃H or PO₃H.

EXAMPLE 9 Synthesis of2-[Tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid also calledPX-13 or TES Trioleate

To a 250 ml single-neck, round-bottom flask 1.0 g (4.26 mmoles) of2-[tris(hydroxylmethyl)methylamino]-1-ethanesulfonic acid (TES; Aldrich;99% purity) and 25.0 mL of anhydrous dimethyl formamide (DMF) wereadded. The flask contents were then cooled to 0° C. in an ice-waterbath. Oleoyl chloride (Aldrich, technical grade) 5.25 g (17.45 mmoles),was added dropwise over a 5 minute period. The reaction mixture wasstirred at room temperature for 4 days. We have also performed thisreaction by stirring for a shorter period of time at elevatedtemperatures. The DMF was removed at 40-45° C. under reduced pressure.The residue was a viscous oil which was transferred to a flaskcontaining 200 mL acetone and vigorously stirred until a slightly cloudysolution formed. This solution was refrigerated overnight. Theprecipitate formed was collected by filtration and thoroughly extractedwith distilled acetone (20 mL) five times. The product was dried invacuo at room temperature for 24 h producing a yield of 2.32 g (52%),and has the following structural configuration:

Another method of synthesizing 2-[Tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid is provided in the followingparagraph.

To 49.0 g (219 mmol) of2-[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid in 500 mL ofCH₃CN was added 385 mL (990 mmol) of 85% oleoyl chloride. Theorange-brown suspension was stirred under nitrogen at 35° C. for 36 h.Then 500 mL of acetone were added into the brown suspension. Thereaction mixture was put in the freezer overnight. The light brown solidwas reprecipitated from ethanol, then from methanol/acetone(approximately 1:9) and from ethanol again. The white solid was dried invacuo. The yield was 152.2 g (70%) and the melting point was 62-64° C.

EXAMPLE 10 Synthesis of N-[Tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)succinic acid monoamide

This compound is represented by the following structural configuration:

To 566 mg (0.553 mmol) of 2-[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid in 10 mL THF was added 410 mg (4.06mmol) of triethylamine. After 5 min 96 mg (0.960 mmol) of succinicanhydride was added. The yellow solution was stirred for 18 h underreflux. The solvent was removed under reduced pressure. The residual oilwas purified by chromatography on silica gel, eluted with an ethylacetate/methanol gradient (25:75 vol/vol to 0:100 vol/vol).N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl) succinic acid monoamidewas isolated as a yellow heavy viscous oil with a yield of 240 mg (38%).

EXAMPLE 11 Synthesis of N-[Tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)tartaric acid monoamide

Synthesis of N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl) tartaricacid monoamide was accomplished in two steps. First, reaction of2-[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid withdiacetyltartaric acid anhydride resulted inN-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-2,3-diacetyltartaricacid monoamide. This was hydrolyzed with sodium hydroxide to yieldN-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl) tartaric acidmonoamide. The reaction is described in detail below.

First the synthesis ofN-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-2,3-diacetyltartaricacid monoamide was performed by adding added 1.23 g (11.6 mmol) oftriethylamine to 2.07 g (2.02 mmol) of2-[tris(oleoyloxymethyl)methylamino]-1-ethanesulfonic acid in 150 mLTHF. After 5 min 793 mg (3.66 mmol) of diacetyltartaric anhydride wasadded. The yellow solution was stirred for 18 h under reflux. Thesolvent was removed under reduced pressure. The residual oil waspurified by chromatography on silica gel, eluted with an ethylacetate/methanol gradient (20:80 vol/vol to 0:100 vol/vol) and a traceof acetic acid.N-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-2,3-diacetyltartaricacid monoamide was isolated as a yellow heavy viscous oil with a yield1.36 g (56%).

Next, to 1.30 g (1.05 mmol) ofN-[tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl)-2,3-diacetyltartaricacid monoamide in 50 mL THF was added 20 mL 0.5 aqueous sodiumbicarbonate. After 60 min treatment with ultrasound the solvent wasremoved under reduced pressure. The residual mixture was suspended inwater and extracted with ether.N-[Tris(oleoyloxymethyl)methyl]-N-(2-sulfoethyl) tartaric acid monoamidewas isolated as a yellow heavy viscous oil with a yield of 1.12 g (92%).

EXAMPLE 12 Tris(oleoyloxymethyl)methylamine p-Toluene Sulfonic Acid

Tris(hydroxymethyl) aminomethane (Tris, Aldrich) 0.54 g (4.4 mmoles),5.0 g (17.7 mmoles) of oleic acid (Aldrich), and 1.26 g (6.6 mmoles) ofp-toluenesulfonic acid monohydrate (Sigma) were mixed in 50 mL oftoluene and placed in a 100 mL round-bottomed single-necked flaskequipped with a Dean-Stark trap and a Teflon-coated magnetic stirringbar. After bubbling the reaction mixture with N₂ gas for 10 minutes, thereaction mixture was stirred and heated to reflux. The reaction wascontinued until a stoichiometric amount of water was recovered (0.38ml). After removal of a small amount of undissolved material byfiltration, the toluene was removed by rotoevaporation to yield a whitewaxy product. This product was purified on a silica gel column (Aldrich230-400 mesh, 2.0×55 cm) with 8:2 petroleum ether (bp 60-90° C.)—ethylacetate as developing solvent. After eluting with developing solvent thetop uncolored layer was carefully removed and the product extracted withethyl acetate from the silica gel. The solvent was removed byrotoevaporation and the product recovered as atris(oleoyloxymethyl)methylamine p-toluene sulfonic acid having thefollowing structural configuration:

In this procedure, the amine group of the Tris is protected fromreacting with the fatty acid because it is in the form of a p-toluenesulfonate salt. Moreover, the p-toluene sulfonic acid acts as a catalystfor the esterification between the alcohol functions on the Tris and thefatty acid.

EXAMPLE 13 Generic Formula for PX-18 Related Compounds

These compounds are represented by the following structuralconfiguration:

A comprises H, OH, a sugar moiety, an ether, an ester, an amide or NH₂,or an acid group or salt thereof. Some of the preferred acids includeCOOH, SO₃H, and PO₃H.

B is N, NR, P, P═O, CH or CR wherein R is an alkyl chain of 1 to 6carbons which may be functionalized or non-functionalized.

C₁, C₂ and C₃ are connecting groups selected from the group consistingof —(CH₂)_(n)— wherein n is an integer from 1 to 24, and the —(CH₂)_(n)—chain may be functionalized or non-functionalized. C₁, C₂ and C₃ mayalso be selected from the group consisting of poly(ethylene oxide) ofthe formula (CH₂CH₂—O)y wherein y is an integer from 1 to 12. C₁, C₂ andC₃ may be the same or different.

D₁ and D₂ are fatty acid chains consisting of fatty acid esters of theform CH₃(CH₂)_(n) COO or fatty acid amides of the form CH₃(CH₂)_(n) CONHwherein n is an integer from 1 to 32. At least one of the fatty chainsis unsaturated at one or more positions, and D₁and D₂ may be the same ordifferent with respect to length and degree of unsaturation.

In the generic formula listed above, the fatty acid chains of themolecule may be comprised of (CH₂)_(n) wherein n is an integer from 1 to32. These fatty acid chains may each be unsaturated at one or more sitesand may be of different lengths from 1 to 32 carbon atoms.

EXAMPLE 14 BES Dioleate, also called PX-18 or2-[N,N-Bis(2-oleoyloxyethyl)amino]-1-ethanesulfonic acid

A preferred embodiment of the generic structure in the preceding Exampleis represented by the following structural configuration:

In the formula for PX-18 shown above, the SO₃H acid group may also be inthe saltform or in an ionized form.

N,N-Bis(2-hydroxyethyl)-2-aminoethane sulfonic acid was prepared(according to Izumi, 1954) by refluxing an aqueous solution of sodium2-bromoethanesulfonate with 2 equivalents of diethanolamine for 2 hours.The cooled reaction mixture was passed over a sulfonic acid resin (Dowex50) in the acid form. The eluate, containing product and HBr, was takento dryness at reduced pressure and the product was recrystallized fromaqueous alcohol. The yield was 52% and the compound had a melting pointof 153-155° C.

In a 1 liter round bottomed flask, 16.2 g (0.076 mol) ofN,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid were dissolved in asolution of 60 mL of DMF and 36 mL of triethyl amine. To this solution72 g (0.239 mol) of oleoyl chloride (85% Aldrich) were slowly addedunder a N₂ atmosphere with stirring. (During the addition of the oleoylchloride a precipitate is produced). After all of the oleoyl chloridewas added, 300 mL of acetone or THF was added to the mixture. Thereaction mixture was allowed to stir under a N₂ atmosphere for 12 h. Theprecipitate was collected by a filtration, and then recrystallized fromCHCl₃/CH₃ CN. A yellowish product (43.6 g) was obtained in 77% yield.The product was decolorized with charcoal in CH₃Cl, recrystallized fromCHCl₃/CH₃CN, and exhibited a melting point of 133-136° C.

EXAMPLE 15 Topical Cream Formula and Formulation Including TES Trioleate(4% PX-13)

Ingredients for solution A are 8 g cetyl alcohol; 2 g oleic acid; 2 gisopropyl myristate 0.5 g paraben; 4 g PX-13. Ingredients for solution Bare 78 ml dH₂0 (78 g); 1 g TRIS.HCl; 3.0 g SDS (sodium lauryl sulfate);3 g sorbitol; 5 g 1,2 propanediol.

Formulation of Skin Cream

Formulation of solution A: In a 100 mL beaker cetyl alcohol was heatedto melting (approximately 70° C.) and the oleic acid andisopropylmyristate are added and mixed together with the paraben. Whenthese ingredients are in solution, the PX-13 was added and thetemperature maintained at 70° C. until solution occurred.

Formulation of solution B: In 200 mL beaker the water was brought toboiling and then removed from the hot plate. TRIS.HCl and SDS were addedand stirred until dissolved. The sorbitol and propanediol were thenadded with stirring in succession while maintaining the temperature atapproximately 70.° Next, solution B was rapidly stirred while solution Awas slowly added. The mixture was thoroughly stirred (for about 5 min).While the mixture was hot (70° C.) 25 g was poured into 4 (1 oz)dispensing tubes.

It is understood that the formulation may optionally contain additivessuch as lanolin, aloe, herbal extracts, or various scents such as floralscents. Additionally, the formulation may optionally containpreservatives, antimicrobials, penetration enhancers and other skincream ingredients commonly known to one skilled in the art.

EXAMPLE 16

Dermatologic Disorders

Studies were conducted with skin creams produced under GMP conditionscontaining PX-11 (tricinyl trioleate) and PX-13 and prepared accordingto the following recipe using ingredients known to one of ordinary skillin the art.

TABLE Base Cream Experimental d H₂O 92.5% 87.5% carbopol 0.3 0.3glycerin 2.0 2.0 pollex 2.0 2.0 Finsolv 2.0 2.0 PX-11/13 (active) — 5.0Germaben 2B 1.0 1.0 AMP (amine) 0.2 0.2

These products were used with four psoriatic and three atopic dermatitisvolunteers. PX preparations applied topically to psoriatic lesions twicea day over a three-week period reduced swelling, diameter and elevationof plaques in all patients. In the atopic dermatitis patients there wasalso improvement including reduced erythema, itching and burning.

Evaluation of PX Compounds in Cream Formulations

Two PX compounds PX-11 and PX-13 were evaluated in a cream formulationprepared under GMP. The chemicals were formulated into a bland cream andevaluated in the following human or mouse skin tests: (1) occluded patchtest for irritation; (2) cell turnover rate; (3) transepidermal waterloss; (4) skin moisturization; and (5) reduction in erythema afterinduced sunburn.

The human skin tests were conducted on three volunteer female subjects,ages 29-32; a cream base with no added active was included as a control,while the untreated skin served as an additional basis for evaluation.Sunburn tests were performed on a total of 30 hairless albino mice.Results of the studies are summarized below.

1. Occluded patch test. Samples were applied on the subjects' backsbetween the scapulae and waist adjacent to the spinal column. Each sitewas sensitized to the chemicals by removal of half the stratum corneumbarrier by tape stripping. The levels of irritation on the sensitizedsites were measured after 24 hours of occluded exposure to the creams.The levels of erythema, edema, vesiculation and blister formation werescored. All samples were non-irritating.

2. Cell turnover rate: This parameter was measured by adsorption ofdansyl chloride, in which 3% dansyl chloride was incorporated intopetrolatum. This salve was applied over a 2.5 cm diameter circular areain the upper arm and a patch applied. 24 hours later the patch wasremoved. The intensity of dansyl chloride fluorescence was indicative ofthe cell turnover rate in the epidermis.

It was found that the control cream decreased cell regeneration time by35%, i.e., the cells renewed more rapidly compared with the untreatedskin (a skin patch with nothing applied). The addition of PX samplesinhibited the rate of cell regeneration. In fact, for PX-11, the time toregenerate was longer than that observed for the untreated skin (seetable below).

Days to Cell Renewal % Change in Cell Untreated Treated Turnover TimePX-11 21.5 28.8 +34 PX-13 30.4 21.6 −29 Control 30.4 19.7 −35

Note that the control cream accelerates cell growth but in the presenceof PX-11 and PX-13 cell growth is inhibited. For PX-11, growth is soseverely retarded that the treated skin area requires significantlylonger times for the cells to regenerate. These results suggest that PXcompounds ,may be useful in the treatment of psoriasis.

3. Transepidermal water loss. This parameter was measured by affixing aflow cell on the forearm site, applying the cream, waiting 20 minutes,then flushing the volume with nitrogen and noting the moisture contentof the effluent gases. It was found that PX-13 functioned as anoccluding moisturizer, reducing the rate at which water is lost from theepidermal layer. The control and PX-11 opened the pores and permittedwater to escape from the epidermal layer. PX-11 was 10 times moreeffective than the control at promoting water loss.

% Change in Transepidermal Water Loss PX-11 +649.0 PX-13 −8.5 Control+56.5

4. Skin moisturization. Skin moisturization was measured using 20megahertz ultrasound (the presence of moisture produces a densitydecrease in the outer skin layers), before and 30 minutes after productapplication to the forearm. It was found that PX-11 exhibited goodmoisturization properties, while PX-13 and the control were not goodmoisturizers.

Moisturization Effects Ultrasound Density Score Subject PX-11 PX-13Control 1 3 1 2 2 3 2 1 3 1 −1 −2 Total 7 2 1 7 = Best; 1 = Baseline

5.Protection against ultraviolet-induced ervthema. The protectionagainst ultraviolet-induced erythema (i.e., the burn and irritationproduced after exposure to ultraviolet radiation), was measured by firstirradiating 12 mice with destructive 3130 angstrom (Å) rays, applyingthe creams, and then evaluating the skin cells for erythema. PX-11 wassignificantly better than the base cream in erythema reduction.

Post-Radiation Ervthema Rankings Subject PX-11 PX-13 Control 1 2 5 3 2 25 4 3 2 4 5 Mean 2 4.7 4 1 = Best; 4 = Worst

The results presented in Example 16 suggest that the PX test compoundswere active in modifying human skin cell activity and, therefore, mayimpart beneficial effects when used as a skin cream in a variety ofdermatologic and cosmetic conditions.

EXAMPLE 17

Neurological Inflammatory Conditions

While not wanting to be bound by this statement, it is believed thatPLA₂-mediates inflammation and nerve tissue damage in spinal cord injuryand sciatic-nerve inflammation and in a variety of central andperipheral neurological inflammatory conditions. The followingexperiments demonstrate antiinflammatory activity of PX compounds.

1. Activity of PX-13 to Inhibit PLA₂-Induced Inflammation in Mouse PawEdema Tests

Edema was induced by injection of 1 ug of purified human disc PLA₂ intothe mouse footpad. PX-13 was administered either by gavage orintraperitoneally (IP) 60 min prior to enzyme challenge. Forty-five minafter enzyme injection, the animals were sacrificed and the control andinjected paws were removed and weighed to assess edema. The results arepresented in the following table.

PX-13 (mg/kg) Percent Protection 20 (gavage) 33% 40 (gavage) 55% 60(gavage) 63% 40 (IP) 48%

PX-13 displays anti-inflammatory activity in this model whenadministered by gavage or IP.

2. Protective effects of PX-13 in Cultured Rat Dorsal Root GanglionCells Exposed to Snake Venom and Human Disc PLA₂

Primary cultures of rat dorsal root ganglion cells were used. Cells werewashed 3-times with media to remove serum. Then, PX-13 (20 uM) in HEPESbuffer or a buffer control was applied to the cells. After 10 min ofincubation, purified human disc PLA₂ (activity 1 umol/min) was added andthe cells were incubated for an additional 60-90 min. Cells were thenobserved, fixed, and photographed 1.5 hrs after addition of PLA₂.

Cells treated with PX-13 (20 uM) alone or PX-13+PLA₂ treated cells wereindistinguishable from control cultures, whereas, extensive morphologicdamage was induced by PLA₂ alone within 60 min; considerable cellblebbing and loss of texture was noted. PX-13 is cytoprotective in thissystem to a toxic dose of human disc PLA₂. PX-13 also protected againstthe toxicity induced by purified snake venom PLA₂ used in comparableamounts (1-3 umols/min/mg for 60 min).

These and other results support the concept that the high levels ofsecretory PLA₂ in herniated disc conditions can irritate nerves and maycontribute to the generation of low back pain. These results suggest thepotential efficacy of PX compounds in treating these and relatedneurological conditions.

3. Inhibitory Effect of PX-13 on Nucleus Pulposus (NP)-Induced Mouse PawEdema

Human NP-homogenate was prepared according to the following protocol andstored frozen until use. Human vertebral discs were thawed and thetissue pieces were washed twice with isotonic saline. Tissue sampleswere homogenized in a minimum volume of isotonic saline using a Brinkmanhomogenizer. This homogenate was filtered through two layers ofcheesecloth and the resultant filtrate was subjected to Douncehomogenization. This homogenate was again filtered through two layers ofcheesecloth and the filtrate was capable of passing through a 26-gaugeneedle; 50 ul of the filtrate was administered to each mouse paw.

The filtrate contained 1.57 mg/ml of protein and its PLA₂ activity was12.8% hydrolysis of 10 nmols of E.coli phospholipid/10 ul filtrate/30min. incubation at 37° C. Therefore, each mouse paw received 78.5 ug NPfiltrate. This sample of 78,500 ng total protein contained approximately4.3 ng NP PLA₂ protein. By comparison, we use 500-2000 ng of highlypurified PLA₂ to induce edema in this mouse model; and under theseexperimental conditions the maximal edema achieved is 160% of thecontralateral paw minus 4-7% edema due to saline injection alone.

The data are expressed as percent of contralateral paw and as percent ofmaximal edema achieved with purified PLA₂. Fifty ul (78.5 ug) wasinjected into the paw 30 min. after the mice were treated with PX-13 inHEPES administered IP or by gavage at 30 or 60 mg/kg. After 3 hrs,,thepaws were excised and weighed.

Results

Percent Percent of* Percent Edema Maximal Inhibition Saline Control 106± 2.5 — — NP-Homogenate 124 ± 3.0 34% — alone PX-13: IP 30 mg/kg 120 ±2.0 26% 24% 60 mg/kg 115 ± 1.6 17% 50% PX-13: Gavage 30 mg/kg 122 ± 5.6— — 60 mg/kg 118 ± 2.0 22% 36% *calculated as previously described; PLA₂alone = 160% edema - 106% control = 54% (maximal edema).

Considerable variability or overlap is noted in both 30 mg/kg PXsamples; however, at 60 mg/kg both IP and gavage administration of PX-13inhibited inflammation by 50% and 36%, respectively. These resultsindicate that nucleus pulposus extracts are inflammatory, and PX-13protects against inflammation induced by nucleus pulposus extracts.

4. The Lipid Soluble (PX-13) and Water Soluble (PX-18) Both Inhibit thePurified Human Disc Type II, PLA₂

Both PX-13 and PX-18 inhibit the purified human disc type II, PLA₂. PLA₂activity was measured as described in Franson et al., Prostaglandins,Leukotrienes and Essential Fatty Acids, 43:63-70, 1991, which is hereinincorporated by reference in its entirety. The water soluble compoundPX-18, displayed an IC₅₀ of 0.3 μM whereas the lipid soluble compoundPX-13 displayed an IC₅₀ of 2.5 μM.

EXAMPLE 19

Ischemia-Reperusion Injury

1. Small Bowel Transplantation: Ischemia-Reperfusion Injury

PX-13 and mepacrine inhibited PLA₂ activity in extracts of rat bowel,and PX-13 was a more effective inhibitor (by more than 2 log units) ofPLA₂ enzymatic activity than mepacrine. Both compounds were shown toprotect against ischemia and reperfusion injury following small boweltransplantation. These drugs reduced weight of the tissues after storagefor 24 and 48 hours in the perfusate solution.

These drugs were also tested for their ability to enhance thepreservation of tissues stored for subsequent transplantation. The smallbowel was removed from rats, washed free of luminal contents and thetissue stored in University of Wisconsin (UW) preservative media ±40 uMPX-13 or mepacrine for 48 hrs. The graft was then transplanted into ratswith or without intravenous (IV) infusion of 20 mg/kg PX-13 or mepacrineat the time of transplantation. Upon transplantation, the bowel isimmediately injured as evidenced by tissue darkening within two minutes.With PX-13 or mepacrine added to the media the gross and microscopicinjury to tissue was dramatically reduced. Animals receiving PX-13maintained normal bowel color in contrast to the blackening (necrosis)which occurs within minutes in the absence of drug. Treatment with theanti-malarial, anti-PLA₂ drug, mepacrine, produced some protectionalthough not as great as with PX-13. Both PX-13 and mepacrine inhibitthe peroxidation (malondialdehyde formation) of homogenized rat bowel,an indication that the membrane-stabilizing, PLA2-inhibitors, also haveanti-oxidant activity.

Recent studies completed in our laboratories demonstrate that ischemicbowel releases PLA₂ into the media in a time dependent manner. Theaddition of PX-13 reduced the enzyme release by more than 80% during 24hrs of ischemia.

These results indicate that PX-13, which inhibits PLA₂ activity, exertssignificant protective effects on tissues chosen for transplantation.PX-13 and related compounds may decrease complications resulting fromtissue transplantation thereby increasing successful outcomes andreducing costs. The use of PX-13 and related compounds, acting aspreservation-enhancing additives, may extend storage time and enhancethe preservation of small bowel grafts and other tissue destined fortransplantation in cold storage bathing solutions.

EXAMPLE 20

Parasitic Infection: Protozoal Parasites

1. Malaria: Lipid Soluble PX-13 Inhibits in vitro Replication ofPlasmodium Falciparum

PX-13 was used to block the incorporation of ³H-hypoxanthine into theDNA, to provide a measure of growth, of P.falciparum cultures which wereresistant to the drugs, chloroquine or mefloquine, which are normallyused therapeutically. The growth of two strains in RBC cultures weretested:

The compound PX-13 inhibited the growth of clone W2 (P.falciparum),which is chloroquine resistant and mefloquine sensitive with an IC₅₀ of13 ug/ml. This value for drug resistant clone W2 compares to controlcultures sensitive to both drugs: IC₅₀ chloroquine=30 ng/ml vsmefloquine=1.3 ng/ml.

PX-13 also inhibited the growth of clone D6 (P. falciparum) which ischloroquine sensitive and mefloquine resistant, with an IC₅₀ of 17ug/ml. This value for drug resistant clone D6 compares to controlcultures sensitive to both drugs: IC₅₀ chloroquine=1.6 ng/ml; andmefloquine=4.3 ng/ml.

PX-13 is active against strains of P.falciparum that are resistant tothe drugs currently used for therapy. In addition, the slope of theinhibition curves indicates that PX-13 acts rapidly so that it can morerapidly interfere with the replication process or the disease inprogress. These findings are also significant because the levels ofcurrent therapeutics necessary for control are often toxic. Recentfindings indicate that the therapeutic index of PX-13 (dose for 50%lethality/dose for 50% effectiveness or LD₅₀/ED₅₀) is a large positivenumber, or highly favorable relative to the known toxicity problems forchloroquine, mefloquine, and other drugs used in the treatment ofmalaria.

2. Malaria: Water Soluble PX-18 Inhibits in vitro Replication ofPlasmodium Falciparum

The water soluble compound PX-18 inhibited the same drug-resistant P.falciparum strains as described above. The IC₅₀ for PX-18 was 1.3 and2.4 ug/ml. Thus, the water soluble PX-18 compound appears to be 10-timesmore effective than the lipid soluble compound PX-13.

The inhibitory effects of lipid soluble PX-13 and water soluble PX-18 onthe growth of drug sensitive and drug resistant species of Plasmodiumfalciparum indicate that these PX compounds and possibly related PXcompounds may be effective anti-parasitic drugs.

EXAMPLE 21

Preservation of Whole Blood

The effect of PX-13 and mepacrine on whole blood cell viability weretested as a function of time of storage. The results are summarizedbelow.

Percent Protection LDH Hemoglobin 48 hr 72 hr 48 hr 72 hr A. Cells alone0 0 0 0 B. Cells + PX-13 20 uM 22 15 55 25 C. Cells + PX-13 80 uM 39 4172 54 D. Cells + PX-13 160 uM 78 72 77 80 E. Cells + mepacrine 20 uM 5827 61 45

Heparinized human blood was centrifuged at 400×g and the resultingplasma was removed and replaced with an equal volume of Dulbecco'smedium. The resuspended cells were then incubated at 37° C. for 0-72hrs. in a shaking water bath. At the indicated times, a 0.8 ml aliquotwas removed and centrifuged at 400×g to sediment cells. Appearance oflactate dehydrogenase (LDH) and hemoglobin in the supernate wasindicative of cell injury. PX-13 and mepacrine stabilized cells anddecreased the injury as evidenced by diminished levels of LDH andhemoglobin in the supernate. This is expressed as percent protection.

The addition of snake venom PLA₂ accelerated the release of both LDH andhemoglobin at 24 hrs (not shown).

EXAMPLE 22

Blocking of Thrombin-activated Platelet Aggregation

Applicants previously demonstrated that a 25-μM dose of PX-13 inhibitedplatelet aggregation. Applicants have also observed that PX-13 inhibitedserotonin release by thrombin-stimulated platelets. This observationsupports the contention that the PX compounds of the present inventionmay block release of inflammatory mediators in the coagulation cascade.This observation also supports the use of PX compounds to inhibitthrombin-induced platelet aggregation and to act as anti-clottingagents.

EXAMPLE 23

Treatment for Snake Bite

Animal and human recipients of venomous snake bites require rapidtreatment to alleviate the toxic inflammatory reactions which may belethal. The compositions of the present invention are available in areadily injectable form for administration to the patient throughintramuscular injection. The compositions of the present invention arestable for periods up to 3 months at room temperature.

Low molecular weight PLA₂ is a major toxic component of snake venoms. Invenoms with neurotoxic effects (i.e. cobra venom), this is mediated by aPLA₂ which binds to a neuronal cells. Snake venom injuries have 3components: 1) peripheral and central neurotoxicity (certain venoms), 2)systemic inflammation, including complement activation, and 3) extensivelocal tissue damage, including muscle necrosis and swelling which cancause distal vascular compromise (compartment syndrome). The followinghypothetical example describes the treatment of a rattlesnake biteoccurring several hours before conventional medical treatment with anemergency snakebite kit containing a water-soluble PLA₂ antagonist,PX-18, in injectable form.

A patient is bit by a rattlesnake on the upper calf while backpackingabove the tree-line in Colorado. He uses his snake bite kit to attemptlocal suction extraction of venom at the bite site. He applies atourniquet proximal to the bite. He then takes out the 2 ml syringe with22 gauge needle, preloaded with 200 mg of PX-18 in sterile solution,which is contained in the kit. Following the kit instructions, heinjects 1 ml of PX-18 intramuscularly (im) for systemic absorption, inthe anterior thigh. He then injects the remaining 1 ml deepsubcutaneously at the bite site, to attempt to neutralize the localconcentration of venom PLA₂. After 30 minutes, he releases thetourniquet and proceeds to seek medical attention.

EXAMPLE 24

Reflex Sympathetic Dystrophy (RSD)

A patient presents with severe RSD characterized by persistent pain andlimitation of movement at the wrist joint. Topical application of PX-13to the wrist in a concentration of about 4% in the cream formulationdescribed in Example 15 results in immediate pain relief and increasedrange of motion within 10 to 15 minutes.

Another patient presents with a two year history of RSD in the kneejoint. This male patient previously received arthroscopic evaluations.This patient also had limited range of motion and became addicted tonarcotics in an attempt to reduce pain. A combination of sympatheticblockade and cream containing PX-13 is topically applied and decreasesthe patient's report of pain and provides a full range of motion in theknee joint. The reduction in pain is immediate and the increased rangeof motion occurs within 10-20 minutes. This patient, who had beenunemployed for an extended period, eliminated intake of narcotics andreturned to work.

A third patient presented with RSD of the foot and ankle. This patienthad no eversion or inversion of the foot. Topical application of PX-13(about 4%) in a cream vehicle provides immediate and complete painrelief and restores full eversion and inversion within 20 minutes.

In each of the cases described above, pain relief analgesia is immediatewith no production of anesthesia.

EXAMPLE 25

Acute Toxicity Studies

An LD₅₀ study was conducted to determine a lethal dose for PX-13suspended in HEPES buffer at a pH of 7.4. A single IP dose of 50, 100,200 or 400 mg/kg was administered to mice (n=6 per group). No deaths orgross physical or behavorial abnormalities were noted after 48 hours inall dosage groups. At 144 hrs one animal in the 400 mg/kg group died. Nonecropsy was performed. The scientist conducting this trial noted that“PX-13 was extremely well tolerated”, confirming the results in the gutreperfusion experiments described in another example following IVadministration of the test compound.

The results indicate that doses of 50 to 200 mg/kg administered IP tomice do not produce acute toxicity in terms of death or gross physicalor behavorial abnormalities.

EXAMPLE 26

Effects of PX-18 on Histamine Release from Basophils

The protocol to evaluate the effects of PX-18 on histamine release isdescribed below. Freshly drawn heparinized blood from donorsnon-medicated for 3 days is diluted 1:5 with PBS containing 1% humanserum albumin (diluent). Test drugs are diluted in diluent to 10 timesfinal concentration, and 25 μl per well is added to duplicate wells ofround-bottomed microtiter plates. Generally 3-fold dilutions of drug areexamined four or five times. 200 μl of diluted blood is then added toeach well. 5 minutes later, 25 μg of rabbit anti-human IgE (purchasedfrom Dako) is added per well, to give final concentrations of 1/400 and1/1200 of antibody. Unstimulated (background release wells) have 25 μlof diluent added. Plates are gently tapped for mixing, then incubatedfor 30 minutes. After incubation, plates are cooled on ice, spun down ina refrigerated centrifuge, and replaced on ice. 100 μl plasma is removedfrom each well for assay, and placed in a 1.5 ml Eppendorf tubecontaining acetylation reagent. Following acetylation, the histamineadduct is quantitated by a sensitive and specific commercial competitiveassay, utilizing the competition of enzyme conjugated acetyl-histaminewith sample analyte for binding to a specific antibody on the solidphase. A sample of blood is also lysed by repeated freeze/thaw todetermine total blood histamine. Results are expressed as a percentageof total blood histamine release. In an acceptable assay maximal releaseis greater than 45% and background release is less than 15%. Experimentsare performed in duplicate or triplicate using separate blood donors.

These experiments demonstrate that PX-18 inhibits the degranulation ofbasophils (and probably mast cells) in response to stimuli such asantibody surface-bound IgE. PX-18 is equipotent to epinephrine, a potentanti-anaphylactic drug, and more efficacious than cromolyn, a prototypic“mast-cell stabilizer”. These results support the use of PX-18 andrelated compounds as compounds to treat allergic disease.

EXAMPLE 27

Effects of PX-18 on Production of Leukotrienes and Prostaglandins byBlood Under Basal and Stimulated Conditions

The following protocol was employed to evaluate effects of PX-18 onproduction of leukotrienes and prostaglandins by blood cells under basalconditions, with ionophore stimulation of cyclooxygenase type I, andwith lipopolysaccharide (LPS) induction of cyclooxygenase type II.Freshly-obtained heparinized blood from medication-free donors isdiluted 1:1 with RPMI, and aliquoted in 0.6 ml amounts in sterilepolypropylene 12×75 ml tubes. Test drugs are added at appropriateconcentrations and diluted in saline with 1% human serum albumin.Control tubes have only diluent or drug vehicle added. Drugs are addedfive minutes prior to addition of stimulus, at 100×concentrations, (6 μlvolumes). This is replicated for 3 racks of tubes, A thru C. A has nostimulus added, and has a 30 minute and 6 hr. tube for each drugconcentration. B has 20 uM of the calcium ionophore A23187 added, and isincubated at 37° C. for 30 minutes. C has 5 ug/ml of E. coli strain BortLPS added, and is incubated at 37° C. for 6 hours. At the end each timeperiod, the appropriate tubes are placed on ice, centrifuged, and 0.4 mlof plasma carefully decanted into a tube containing formic acid and BHTto stop all reactions and preserve eicosanoid products. These tubes arethen frozen at −70° C. for up to one week, then applied to Sep-pac minicolumns and extracted into methanol. The methanol extract is aliquotedinto 3 tubes and evaporated, then stored at −70° C. until the time ofeicosanoid assay. Measurements include thromboxane (by a sensitivefluorometric plate-format assay) and either 15 HETE or LTB4 to measurelipoxygenase products.

The whole blood eicosanoid release experiments show that PX-18 appearsto strongly inhibit the cytokine-inducible cyclooxygenase II, with lessinhibition of the constitutive cyclooxygenase I enzyme, stimulated byionophore, and even less or no inhibition of basal production ofprostaglandins. This is an excellent anti-inflammatory profile as mostNSAID-associated toxicities (i.e., gastric, renal, fluid retention,possibly asthma) are due to inhibition of constitutive cyclooxygenase Iprostaglandin production, which plays a necessary physiologic role.cyclooxygenase II is involved in pathologic inflammation.

These studies also demonstrate that production of lipoxygenase productssuch as LTC4, is inhibited by PX-18. The comparison is to MK 592, aselective lipoxygenase inhibitor. PX-18 does not inhibit either thelipoxygenase or cycloxygenase enzymes, but rather PLA₂ which producesfree arachidonic acid, thus depleting the pool of this enzyme substratefor the lipoxygenase and cyclooxygenase pathways. Our currentexperiments demonstrate that if exogenous arachidonic acid is added andlipoxygenase and cyclooxygenase metabolites are measured, the additionof PX-18, unlike indocin or MK 592 does not inhibit their production.These results support the notion that PX-18 inhibits PLA₂ and notdownstream enzymes such as lipoxygenase and cyclooxygenase.

Additionally, blockade of secretory (s) PLA₂ activity decreases theproduction of lysophospholipid, the immediate and rate-limitingprecursor for platelet-activating factor (PAF). Secretory PLA₂ asopposed to cytosolic perinuclear membrane-localized PLA₂, seems to bethe key enzyme providing substrate which is acetylated to form PAF. PAFis a potent inflammatory mediator, a potent neutrophil chemotactic, anda key factor in tissue injury in such situations as ischemia. PAF isimplicated in inflammatory diseases such as ulcerative colitis, and isunaffected by therapy with NSAIDs.

The invention has been described in detail with particular reference tocertain embodiments, but variations and modifications can be madewithout departing from the spirit and the scope of the present inventionas defined in the appended claims.

What is claimed is:
 1. A composition comprising:

wherein A comprises H, OH, a sugar moiety, an ether, an ester, an amide,NH₂, or an acid or salt thereof; B is selected from the group consistingof N. NR, P, P═O, CH, and CR, wherein R is an alkyl chain of 1 to 6carbons and the chain may be functionalized or non-functionalized; C₁,C₂ and C₃ are connecting groups which may be different and are selectedfrom the group consisting of —(CH₂)_(n)— and (CH₂CH₂—O)y wherein n is aninteger from 1 to 24, —(CH₂)_(n)— may be functionalized ornon-functionalized, and y is an integer from 1 to 12; and D₁ and D₂ arefatty acid chains which may be different and are selected from the groupconsisting of fatty acid esters of the form CH₃(CH₂)_(n) COO, and fattyacid amides of the form CH₃(CH₂)_(n) CONH, wherein n is an integer from1 to 32, at least one of the fatty chains is cis-unsaturated at one ormore positions, and the fatty chains may be of different lengths and maybe unsaturated at different locations with the proviso that when B is N,NR, or CR, that A is not H or OH when C₃ is —(CH₂)_(n)—.
 2. Thecomposition of claim 1, wherein A comprises H, OH, NH_(2,) or an acidselected from the group consisting of COOH, SO₃H, and PO₃H, or saltthereof, and n is an integer from 1 to
 10. 3. The composition of claim1, wherein D₁, and D₂ are fatty acid esters of the form CH₃(CH₂)_(n)COOand n is an integer between 1 and
 24. 4. A composition comprising:

wherein A comprises a sugar moiety, an ether, an ester, an amide, NH₂,an acid or salt thereof; B is selected from the group consisting of N,NR, P, P═O, CH, and CR, wherein R is an alkyl chain of 1 to 6 carbonsand the chain may be functionalized or non-functionalized; C₁, C₂ and C₃are connecting groups which may be different and are selected from thegroup consisting of —(CH₂)_(n)— and (CH₂CH₂—O)y wherein n is an integerfrom 1 to 24, —(CH₂)_(n)— may be functionalized or non-functionalized,and y is an integer from 1 to 12; and D₁ and D₂ are fatty acid chainswhich may be different and are selected from the group consisting offatty acid esters of the form CH₃(CH₂)_(n)COO, and fatty acid amides ofthe form CH3(CH2)_(n)CONH, wherein n is an integer from 1 to 32, atleast one of the fatty chains is cis-unsaturated at one or morepositions, and the fatty chains may be of different lengths and may beunsaturated at different locations.
 5. The composition of claim 4,wherein A is an acid selected from the group consisting of COOH, SO₃H,and PO₃H, or salt thereof, and n is an integer from 1 to
 10. 6. Thecomposition of claim 4, wherein D₁ and D₂ are fatty acid esters of theform CH₃(CH₂)_(n)COO and n is an integer between 1 and
 24. 7. Thecomposition of claim 4, wherein A is a salt of an acid.
 8. A compositioncomprising:

wherein A comprises H, OH, a sugar moiety, an ether, an ester, an amide,NH₂, an acid or salt thereof; B is selected from the group consisting ofP, P═O, and CH; C₁, C₂ and C₃ are connecting groups which may bedifferent and are selected from the group consisting of —(CH₂)_(n)— and(CH₂CH₂—O)y wherein n is an integer from 1 to 24, —(CH₂)_(n)— may befunctionalized or non-functionalized, and y is an integer from 1 to 12;and D₁ and D₂ are fatty acid chains which may be different and areselected from the group consisting of fatty acid esters of the formCH₃(CH₂)_(n)COO, and fatty acid amides of the form CH₃(CH₂)_(n)CONH,wherein n is an integer from 1 to 32, at least one of the fatty chainsis cis-unsaturated at one or more positions, and the fatty chains may beof different lengths and may be unsaturated at different locations. 9.The composition of claim 8, wherein A is an acid selected from the groupconsisting of COOH, SO₃H, and PO₃H, or salt thereof, and n is an integerfrom 1 to
 10. 10. The composition of claim 8, wherein D₁ and D₂ arefatty acid ester of the form CH₃(CH₂)_(n)COO and n is an integer between1 and
 24. 11. A composition comprising:

or a salt thereof.
 12. A composition comprising:

wherein A comprises H, OH, a sugar moiety, an ether, an ester, an amide,NH₂, or an acid or salt thereof and n is an integer from 1 to
 24. 13.The composition of claim 12, wherein A comprises H, OH, NH₂, or an acidselected from the group consisting of COOH, SO₃H, and PO₃H, or saltthereof, and n is an integer from 1 to
 10. 14. The composition of claim12, wherein A is a salt of an acid.
 15. The composition of claim 12,wherein A is an acid selected from the group consisting of COOH, SO₃H,and PO₃H.
 16. The composition of claim 12, comprising

or a salt thereof.
 17. The composition of claim 12, comprising

or a salt thereof.
 18. The composition of claim 12, comprising

or a salt thereof.
 19. The composition of claim 12, comprising

or a salt thereof.
 20. The composition of claim 12, comprising

or a salt thereof.